RHODOSPIN GENE CORRECTION BY OLIGONUCLEOTIDE TARGETING

描述（改编自申请人的摘要）：三重定向和其他 将测试基于多核苷酸的战略的可行性， 常染色体显性视网膜色素变性（ADRP）的基因特异性治疗 是由视紫红质基因缺陷引起的在这项工作中， 研究人员将开发可用于测试各种 ADRP和其他常染色体显性遗传病的治疗方法 紊乱由于ADRP的任何疗法最终都必须在临床试验中进行测试和优化， 动物，研究人员选择了小鼠，因为他们的容易， 基因组可以被修改。研究者建议建立一个小鼠ES细胞 DNA的线选择片段，可以有效地靶向 位点特异性重组。这些修饰的ES细胞将允许高效， 任何形式的视紫红质、cDNA或基因组、野生型或 突变体，来自任何物种，用于广泛的生理学研究。的 研究人员将“敲入”经过修饰的人类视紫红质基因， 作为寡核苷酸处理对细胞的影响的灵敏细胞检测器， 视紫红质基因表达、校正和敲除。对于整个小鼠研究， 研究者将使用视紫红质与绿色荧光蛋白融合物 （GFP）。寡核苷酸对转录的影响将被测量为减少 寡核苷酸对基因校正的影响， 突变将通过GFP荧光的出现来测量， 不表达的视紫红质-GFP融合构建体（GFP-）。细胞研究 将在整个动物实验之前，以测试各种治疗方法， 参数尽管阿托伐他汀介导的转录作用可能是 容易测量，基于重组的校正和突变的检测 敲除需要专门的结构，研究人员最初将 CHO中选择性腺苷磷酸核糖转移酶（APRT）基因的检测 细胞一个灵敏的检测系统将使我们能够检测到微弱的信号， 然后可以进行改进和优化。使用CHO寡核苷酸（TFO），RNA/DNA 嵌合寡核苷酸和寡核苷酸类似物 酸（PNA），因为它们能够抑制转录，纠正基因缺陷， 并引入突变。这些结果将作为开发的基础。 治疗方案。

DESCRIPTION (Adapted from the applicant's abstract): Triplex-directed and other oligonucleotide-based strategies will be tested for feasibility as gene-specific therapies for autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. In the course of this work the investigator will develop technologies that can be used for testing a variety of therapeutic approaches to ADRP and other autosomal dominant genetic disorders. As any therapy for ADRP must ultimately be tested and optimized in animals, the investigator has chosen mice because of the ease with which their genomes can be modified. The investigator proposes to create a mouse ES cell line selectable segment of DNA that can be efficiently targeted by site-specific recombination. These modified ES cells will permit efficient, selectable 'knock-in' of any form of rhodopsin, cDNA or genomic, wild-type or mutant, from any species, for a broad range of physiological studies. The investigator will "knock-in" modified human rhodopsin genes, designed to serve as sensitive cellular detectors of the effects of oligonucleotide treatments on rhodopsin gene expression, correction and knockout. For whole mouse studies, the investigator will use fusions of rhodopsin with green fluorescent protein (GFP). Oligonucleotide effects on transcription will be measured as a decrease in fluorescence intensity; oligonucleotide effects on gene correction and mutation will be measured by the appearance of GFP fluorescence starting with rhodopsin-GFP fusion constructs that are not expressed (GFP-). Cellular studies will precede whole animal experiments in order to test various treatment parameters. Although oligonucleotide-mediated effects on transcription can be measured readily, detection of recombination-based correction and mutational knockout requires specialized constructs, which the investigator will initially test at the selectable adenosine phosphoribosyl transferase (APRT) gene in CHO cells. A sensitive detection system will allow us to detect weak signals that can then be improved and optimized. Using CHO oligonucleotides (TFOs), RNA/DNA chimeric oligonucleotides and oligonucleotide analogues such as peptide nucleic acids (PNAs), for their ability to inhibit transcription, correct gene defects, and introduce mutations. These results will be used as the basis for developing treatment protocols in mice.